As part of an ongoing study concerned with improving pneumococcal vaccine, the genes for two defined pneumolysin toxoids (pneumolysoids), Pd-A (with a Cys--Gly substitution at amino acid 428) and Pd-B (with a Try--Phe substitution at position 433), were inserted into a pKK233-2 vector in E. coli and the expressed pneumolysoids were purified. Groups of mice which had been immunized with either pneumolysoid or the native toxin were challenged with virulent pneumococci. Mice in all immunized groups survived significantly longer than sham-immunized controls. Both pneumolysoids were more effective than pneumolysin as protective immunogens. Pneumolysoid Pd-B was conjugated covalently with type 19F PS and the antibody responses of mice were determined to both the protein and the PS moieties of the conjugate. Significant anti-pneumolysin titers were obtained, and the immunogenicity of the 19F PS moiety was markedly enhanced compared with that of unconjugated PS. Conjugation also converted the 19F PS into an antigen capable of inducing a booster effect. These results support the notion that efficacy of pneumococcal vaccine can be improved by addition of pneumolysoid in the form of a covalent PS-pneumolysoid conjugate.